The proposed research is directed towards understanding at a molecular level how chromosomes are moved to daughter cells during cell division by the mitotic spindle. The specific aim of this grant is to determine how the polymerization of microtubules at their site of attachment to chromosomes (the kinetochore) is regulated, and how polymerization and depolymerization at the kinetochore contribute to chromosome movement. Kinetic and structural methods will be used to probe the difference between polymerizing and depolymerizing microtubule ends and how they spontaneously interconvert (dynamic instability). Models which postulate that growing microtubules are stabilized by a cap of GTP containing subunits will be tested by following GTP hydrolysis during polymerization, and examining the effects of non-hydrolyseable analogues on depolymerization rate. The modification of inherent microtubule dynamic behavior which occurs after their capture by kinetochores will be studied in vitro using immunofluorescent and real-time assays. These experiments will examine the role of ATP hydrolysis in microtubule polymerization at the kinetochore and test whether kinetochores can be pulled polewards by depolymerizing microtubules. Functional in vitro assays will be complemented by probing microtubule dynamics in living mitotic cells. A novel photoactivable fluorescent probe attached to tubulin will be used to assess the contribution of microtubule polymerization at the kinetochore to chromosome congression during prometaphase, and the extent to which anaphase movement occurs by depolymerization there. Biochemical methods will be used to bring the analysis of kinetochore function to a molecular level. Cross-linking to tubulin, microtubule affinity, ATP photoaffinity labeling, and subfractionation will be used to identify and characterize the molecules in the kinetochores of isolated mitotic chromosomes responsible for interaction with microtubules. Understanding microtubule dynamics at the kinetochore and its role in chromosome movement may lead to better methods for interfering with the cell-division of tumor cells and parasites, and to the construction of artificial chromosomes for genetic manipulation of food-animals and crops.